A hallmark of cancer and other diseases are high rates of mutation and genome instability, which are believed to be the driving force for disease causing genetic changes. The DMA damage checkpoint is important for the suppression of mutations and genome instabilities, and the regulation of many cellular responses to DMA damage. While an increasing number of genes have been identified to function in the DMA damage checkpoint in human and mutations in some checkpoint genes are linked to cancers, fundamental questions of how the DNA damage checkpoint is regulated remain far from being understood. An important reason for using model experimental systems is to rapidly obtain experimental insights into biological problems that can be applied to problems of human diseases, especially for highly conserved pathways like the DNA damage checkpoint. Yeast, in particular, offers unparalleled advantages in yeast genetics and short generation time. Basic studies in yeast have contributed significantly to the understanding of the DNA damage checkpoint. In the proposed basic studies, we are primarily interested in two basic mechanistic questions about the DNA damage checkpoint. First, how is the DNA damage checkpoint activated? Second, how are various DNA damage checkpoint pathways linked to various DNA damage responses? Accordingly, there are three complementary aims in this proposal. First, we will examine the genetic pathways for Rad53 and Dun1 activation using SmM as a substrate. Second, we will dissect the role of Dun1 phosphorylation in its activation and characterize the roles of Dun1 phosphorylation in the functions of Dun1, including the regulation of dNTP levels, telomere maintenance and suppression of gross chromosome rearrangements. Third, we will use a newly developed Rad53- Dun1-Sml1 system to dissect how Mec1, Tell and Rad53 are activated via biochemical reconstitution and genetic analysis. We will use purified proteins to reconstitute the activation of the Rad53-Dun1-Sml1 system, and further use this system to study the regulation of Mec1 and Tell. Our long-term goal is to understand the regulation of the DNA damage checkpoint kinases and their functions in the regulation of cell growth and suppression of genome instability.